CN110628860A - 植物甾醇转化分离9α-OH-AD和甲酯物的方法 - Google Patents
植物甾醇转化分离9α-OH-AD和甲酯物的方法 Download PDFInfo
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Abstract
本发明公开了植物甾醇转化分离9α‑OH‑AD和甲酯物的方法,属于甾族化合物制备技术领域。发明内容菌株培养、转化产物萃取、9α‑OH‑AD分离提纯、甲酯物分离以及甲酯物提取步骤。本发明以植物甾醇为原料生产9α‑OH‑AD和甲酯物,原料易得,降低了生产成本;本发明还摒弃了传统的以AD作为杂质进行分离纯化的观念,选择合适的萃取剂及萃取条件,采用新的下游分离提取工艺路线,同时获得甲酯物和9α‑OH‑AD两种重要的甾体化合物作为精品,从而提高了提取收率和纯度,降低了生产成本。
Description
技术领域
本发明属于甾族化合物制备技术领域,具体涉及一种植物甾醇转化分离9α-OH-AD和甲酯物的方法。
背景技术
甾体激素药物作为抗生素之后的第二大类药物,对机体起着非常重要的调节作用,甾体微生物转化过程是一个复杂的酶催化过程,涉及侧键降解,上羟基,脱氢等多个反应过程,代谢复杂,难于调控,往往出现一个或多个代谢产物。在以植物甾醇为原料微生物发酵生产9α-OH-AD的过程中,除了甾体水溶性差、转化速度慢以外,代谢产物复杂,分离提取难度大AD等副产物的出现为产品的分离纯化增加了难度,从而降低了底物的转化率和收率,增加生产成本。
英国专利GB1530730公开的以亚硝基胍诱变处理偶发分枝杆菌ATCC6842选育出具有9α-羟基化酶的突变菌株偶发分枝杆菌(Mycobacterium fortuitumNRRL)B-8119,该突变菌株能够转化谷甾醇、胆固醇或豆甾醇生成9α-OH-AD,但转化周期长达336小时,且底物转化率极低,只能得到微量产物9α-OH-AD。
发明内容
针对以上技术问题,本发明的目的是提供一种提高9α-OH-AD和甲酯物分离纯化效率,易操作的植物甾醇转化分离9α-OH-AD和甲酯物的方法。为了解决上述技术问题,本发明所采用的技术方案如下:
本发明提供一种植物甾醇转化分离9α-OH-AD和甲酯物的方法,其特征在于包括以下步骤:
(1)菌株培养:将分枝杆菌(Mycobacterium sp.)B-NRRL 3683诱变菌种经过斜面培养和种子培养后接种至转化培养基中,培养转化后得到发酵液;
(2)转化产物萃取:将所述发酵液加入氯仿,氯仿层减压浓缩,油层加入甲醇搅拌萃取,静置,分出上层甲醇,下层油层用甲醇再萃取2次;
(3)9α-OH-AD分离提纯:将甲醇层合并,减压浓缩至糊状,加水升温后继续减压浓缩,带干其中的甲醇,然后冷却养晶,抽滤得到混合物1,所述混合物1加入甲苯后升温打浆,降温后抽滤,得甲苯母液层和滤饼,滤饼用少量甲苯淋洗后烘干,得9α-OH-AD精品;
(4)甲酯物分离:将所述甲苯母液层升温减压浓缩至糊状,加水带干其中的溶剂,降温,调pH至12以上,抽滤,得甲酯物粗品。
(5)甲酯物提取:将所述甲酯物粗品加入有机溶剂,升温60℃回流打浆,降温、过滤、烘干得甲酯物精品。
优选地,在步骤(1)中所述斜面培养基组分为:在步骤(1)中所述斜面培养基组分为:蛋白胨0.1-10g/L,酵母膏0.1-10g/L,葡萄糖0.1-10g/L,磷酸氢二钠0.1-10g/L,琼脂20g/L,pH=7.5-8.0。
优选地,在步骤(1)中所述液体种子培养基组分如下:柠檬酸1-2g/L,甘油10-20g/L,柠檬酸铁铵0.01-0.05g/L,七水硫酸镁1-1.5g/L,硝酸钠1-2g/L,磷酸氢二铵1-3g/L,玉米浆干粉1-10g/L;pH7.1-7.2,在121℃下高压蒸汽灭菌30min。
进一步地,步骤(1)中所述发酵培养基组分如下:玉米浆干粉10-20g/L,磷酸二氢钾1-10g/L,硝酸钠1-10g/L,硫酸铵1-10g/L,硫酸锌0.01-0.1g/L,硫酸镁0.1-1g/L,消泡剂1-10g/L,豆油100-200g/L,甾醇10-100g/L;pH7.5。
优选地,所述步骤(1)中的生物转化方法具体为:按所述配方配制发酵培养基,121℃灭菌30min,冷却至30℃左右,于无菌条件下接种质量百分比为10-20%的分枝杆菌种子液;转化条件:于28-32℃,空气流量0.5-1.0vvm,罐压0.05-0.06MPa下转化。
优选地,所述步骤(2)中转化产物的萃取方法具体为:在所述转化产物中加入氯仿搅拌萃取1h,氯仿层减压浓缩;油层加入3L甲醇,常温搅拌萃取1h,静置2h,分出上层甲醇,下层油层用甲醇再萃取2次,甲醇用量为每次3L;
优选地,所述步骤(3)中9α-OH-AD的分离提纯方法具体为:将甲醇层合并,50-70℃减压浓缩。待浓缩至糊状,加水,升温至70℃,继续减压浓缩带干其中的甲醇,冷却至20-30℃,养晶1小时,抽滤得到混合物1;将所述混合物1烘干,加入2V甲苯,升温至70℃-80℃,打浆2-4小时,降温到20-30℃进行抽滤,得甲苯母液层和滤饼,滤饼用少量甲苯淋洗,于70℃烘干,得9α-OH-AD精品。
优选地,所述步骤(4)甲酯物的分离方法具体为:将所述甲苯母液层升温减压浓缩至糊状,加入适量水带干其中的溶剂,降温到20-30℃,加入氢氧化钠调pH至12以上,抽滤,得甲酯物粗品。
优选地,所述步骤(5)甲酯物的提取方法具体为:将所述甲酯物粗品加入10V质量浓度为50%的有机溶剂,升温60℃回流打浆1小时,降温到30℃以下,过滤得白色固体,于70℃烘干,得甲酯物精品。
优选地,所述有机溶剂为甲醇、丙酮、乙醇中的任意一种。
本发明的有益效果在于:
1.本发明以植物甾醇为原料生产9α-OH-AD和甲酯物,原料易得,降低了生产成本。
2.本发明摒弃了传统的以AD作为杂质进行分离纯化的观念,选择合适的萃取剂及萃取条件,采用新的下游分离提取工艺路线,同时获得甲酯物和9α-OH-AD两种重要的甾体化合物作为精品,从而提高了提取收率和纯度,降低了生产成本。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
以下通过实施例对本发明作进一步阐述,但不作为对本发明的限定。
实施例1菌种诱变
出发菌种:Mycobacterium sp.B-NRRL 3683
(1)菌种培养:
固体斜面培养基:M1+2%琼脂。
液体种子培养基:M1。
在固体斜面培养基中培养菌株B-NRRL 3683,接一环生长良好的斜面种子在装有100ml液体种子培养基的500ml三角瓶中活化,30℃,200rpm摇床振荡培养48h;取活化好的一级液体种子10ml,接入装有100ml液体种子培养基的500ml三角瓶中进行二级种子培养,30℃,200rpm摇床振荡培养48h。
(2)菌悬液制备
将生长好的二级种子装入10ml离心管中10000rpm离心5min,弃上清液,收集菌体,用ph6.0的磷酸钾缓冲液离心洗涤两次,再用无菌磷酸钾缓冲溶液(pH6.0)制成菌悬液,并将浓度稀释至108-109个/ml。
(3)亚硝基胍(NTG)诱变处理
取上述菌悬液2ml,0.1mol/L亚硝基胍溶液1ml,0.2mol/Lph6.0的磷酸钾缓冲液2ml,加入离心管中,充分混匀,立即置于30℃水浴分别振荡(避光条件下)处理25-35分钟后,离心收集菌体,用PBS冲洗3次以去除NTG残留,最后向离心管中加入5ml无菌生理盐水,摇匀后取出一定的菌悬液,用生理盐水稀释至一定浓度备用。取100ul上述菌悬液涂布到固体平板培养基上,30℃避光培养,并计算致死率。
致死率=(0s时的菌落数-不同诱变时间的菌落数)/0s时的菌落数*100%
经计算得出,25-35分钟平均致死率分别为:65.2%、82.1%、91.7%。当致死率在80%时,细菌诱变效果最好,30min为最佳处理时间。
将30min作为试验中最佳诱变时间,按上述方法对出发菌株的菌悬液进行诱变处理,在1200个单菌落中选出8个生长良好的单菌落,用筛选出的8支突变菌株和出发菌株进行植物甾醇发酵,并检测9α-OH-AD和甲酯物产量,得到目标诱变菌种。
实施例2种子培养
菌种名称:Mycobacterium sp.B-NRRL 3683诱变菌种
1、斜面培养
配方:蛋白胨0.1-10g/L,酵母膏0.1-10g/L,葡萄糖0.1-10g/L,磷酸氢二钠0.1-10g/L,琼脂20g/L,pH=7.5-8.0。
接种后,于29℃培养4-5天,斜面活化2-3次。
2、液体种子培养
配方:柠檬酸1-2g/L,甘油10-20g/L,柠檬酸铁铵0.01-0.05g/L,七水硫酸镁1-1.5g/L,硝酸钠1-2g/L,磷酸氢二铵1-3g/L,玉米浆干粉1-10g/L,pH 7.5。
121℃高压蒸汽灭菌30min。冷却至室温。
一级培养:于无菌条件下接种,用接种环刮取一环菌体接入装有100ml种子培养基的500ml三角瓶中,30℃、200rpm培养至对数生长期(48-64h)。
二级培养:种子培养基同一级种子培养基,接种量为10%,2升种子瓶装样600毫升,培养条件:30℃,200rpm培养至对数生长期(48-64h)。
实施例3发酵转化
(1)种子培养
按照实例2进行种子培养;
(2)转化
在10升发酵罐内进行发酵转化,计料体积6升,接种后体积6升,接种量1.2升。
发酵培养基组分如下:玉米浆干粉10-20g/L,磷酸二氢钾1-10g/L,硝酸钠1-10g/L,硫酸铵1-10g/L,硫酸锌0.01-0.1g/L,硫酸镁0.1-1g/L,消泡剂1-10g/L,豆油100-200g/L,甾醇10-100g/L;pH7.5。
本实施例中所使用的发酵培养基组分具体如下:玉米浆干粉14g/L,磷酸氢二铵1g/L,硝酸钠1g/L,硫酸铵1g/L,硫酸锌0.05g/L,硫酸镁0.1g/L,消泡剂5g/L,植物甾醇50g/L,大豆油160g/L;pH7.5。
在10升发酵罐中加入大豆油,植物甾醇,后加入配制好的发酵培养基,121℃高压蒸汽灭菌30分钟,待冷却至30℃左右,火圈保护下接种1.2升,转化体系终体积为6升,转化条件:30℃、200rpm,空气流量0.05VVM,罐压0.05MPa。转化120小时,植物甾醇转化完全,结束转化。
生物转化的反应式如下所示:
发酵液送液相检测,其中9α-OH-AD含量85.7%,甲酯物含量5.8%。
(3)转化产物的分离纯化
3.1转化产物萃取
在发酵液中加入氯仿搅拌萃取1h,氯仿层减压浓缩;油层加入3L甲醇,常温搅拌萃取1h,静置2h,分出上层甲醇,下层油层用甲醇再萃取2次,甲醇用量为每次3L;
3.2 9α-OH-AD分离提取
将甲醇层合并,50-70℃减压浓缩。待浓缩至糊状,加水,升温至70℃,继续减压浓缩带干其中的甲醇,冷却至20-30℃,养晶1小时,抽滤得到淡黄色固体状的混合物1;将所述混合物1烘干,加入2V甲苯,升温至70℃-80℃,打浆2-4小时,降温到20-30℃进行抽滤,得甲苯母液层和滤饼,滤饼用少量甲苯淋洗,于70℃烘干,得9α-OH-AD精品130.6克,其HPLC归一含量为98.9%。
3.3甲酯物分离
甲苯母液层升温减压浓缩至糊状,加入适量水带干其中的溶剂,降温到20-30℃,加入氢氧化钠调pH至12以上,抽滤,得到黄色固体状的甲酯物粗品41.2克,样品送液相进行归一含量检测,其中甲酯物含量为48.39%,9α-OH-AD含量为34.95%。
3.4甲酯物提取
将步骤3得到的甲酯物粗品,加入10V 50%浓度的甲醇,升温60℃回流打浆1小时,降温到30℃以下,过滤,得白色固体,70℃烘干,得甲酯物精品8.2克,送样HPLC检测,纯度95.2%。
实施例4
(1)按照实施例2的方法进行种子培养。
(2)按照实施例3的方法进行生物转化,发酵液送液相检测,其中9α-OH-AD含量87.5%,甲酯物含量6.2%。
(3)提取及精制
按照实施例3的方法进行提取和精制,得9α-OH-AD精品和甲酯物粗品,其中9α-OH-AD精品的质量为129.3克,HPLC归一含量为98.3%;
将得到的甲酯物粗品加入10V 50%丙酮水溶液,过滤,滤饼70℃烘干,得8.5克甲酯物精品,HPLC归一含量为96.9%。
实施例5
(1)按照实施例2的方法进行种子培养。
(2)按照实施例3的方法进行生物转化;发酵液送液相检测,其中9α-OH-AD含量84.5%,甲酯物含量6.8%。
(3)提取及精制
按照实施例3的方法进行提取和精制,得9α-OH-AD精品和甲酯物粗品,其中9α-OH-AD精品的质量为134.5克,HPLC归一含量为97.8%;
将得到的甲酯物粗品加入10V 50%乙醇水溶液,过滤,滤饼70℃烘干,得9.2克甲酯物精品,HPLC归一含量为96.2%。